ES2655389T3 - Modified compstatin with peptide skeleton and c-terminal modifications - Google Patents

Abstract

A compound comprising a modified compstatin peptide having SEQ ID NO: 1, which is ICVVQDWGHHRCT (cyclic C2-C12), in which the Gly at position 8 is modified to restrict the confirmation of the peptide skeleton at that location , where the skeleton is restricted by replacing the Gly with N-methyl Gly.

Description

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Modified compstatin with peptide skeleton and c-terminal modifications Description

FIELD OF THE INVENTION

This invention relates to the activation of the complement cascade in the body. In particular, this invention provides peptides and peptidomimetics capable of binding with the C3 protein and inhibiting complement activation.

BACKGROUND OF THE INVENTION

The human complement system is a powerful player in the defense against pathogenic organisms and the medication of immune responses. The complement can be activated by three different routes: the classical, lectin, and alternative routes. The main activation event that is shared by the three pathways is the proteolytic cleavage of the core protein of the complement system, C3, in its activation products C3a and C3b by C3 convertases. The generation of these fragments leads to opsonization of pathogenic cells by C3b and iC3b, a process that makes them susceptible to phagocytosis and clearance, and to the activation of immune cells through an interaction with complement receptors (Markiewski & Lambris, 2007, Am J Pathol 171: 715-727). The deposition of C3b in target cells also induces the formation of new convertase complexes and thus initiates a self-amplification loop.

A set of plasma and cell surface binding proteins carefully regulates complement activation to prevent host cells from self-attacking through the complement cascade. However, excessive activation or inappropriate complement regulation can lead to a variety of pathological conditions, ranging from autoimmune to inflammatory diseases Holers, 2003, Clin Immunol 107: 140-51; Markiewski & Lambris, 2007, supra; Ricklin & Lambris, 2007, Nat Biotechnol 25: 1265-75; Sahu et al., 2000, J Immunol 165: 2491-9). The development of therapeutic complement inhibitors is therefore highly desirable. In this context, C- and C3b have emerged as promising targets because their central role in the cascade allows simultaneous inhibition of onset, amplification, and downward activation of complement (Ricklin & Lambris, 2007, supra).

Compstatin was the first complement inhibitor not derived from the host that showed it was capable of blocking all three activation pathways (Sahu et al., 1996, J Immunol 157: 884-91; U.S. Patent 6,319,897). This cyclic tridecapeptide binds with both C3 and C3b and prevents the cleavage of native C3 by C3 convertases. Its high inhibitory efficacy was confirmed by a series of studies using experimental models that pointed to its potential as a therapeutic agent (Fiane et al., 1999a, Xenotransplantation 6: 52-65; Fiane et al., 1999b, Transplant Proc 31: 934 -935; Nilsson et al., 1998 Blood 92: 1661-1667; Ricklin & Lambris, 2008, Adv Exp Med Biol 632: 273-292; Schmidt et al., 2003, JBiomed Mater Res A 66: 491-499; Soulika et al., 2000, Clin Immunol 96: 212221). The progressive optimization of compstatin has produced analogues with improved activity (Ricklin & Lambris, 2008, supra; WO2004 / 026328; WO2007 / 062249). One of these analogues is currently being tested in clinical trials for the treatment of age-related macular degeneration (AMD), the leading cause of blindness in elderly patients in industrialized nations (Coleman et al., 2008, Lancet 372: 1835 -1845; Ricklin & Lambris, 2008, supra). In view of its therapeutic potential in AMD and other diseases, the further optimization of compstatin to achieve even greater efficacy is of considerable importance.

Previous structure-activity studies have identified the cyclic nature of the compstatin peptide and the presence of both a p-turn and a hydrophobic group as key characteristics of the molecule (Morkikis et al., 1998, Protein Sci 7: 619-627; WO99 / 13899; Morikis et al., 2002, J Biol Chem 277: 14942-14953; Ricklin & Lambris, 2008, supra). For example, WO99 / 13899 disclosed that amino acid residues in positions 5 to 8 have a restricted conformation forming a p-Type I turn and stating that the presence of Gly in position 8 was the most favorable for this structure. Other work using compstatin and compstatin analogues for the development of pharmacophore models revealed that replacing Gly8 with Ala eliminated peptide inhibitory activity (Mallik and Morikis, 2005, J Am Chem Soc 127: 10967-10976). It was discovered that hydrophobic residues at positions 4 and 7 were of particular importance, and their modifications with unnatural amino acids generated an analogue with improved activity 264 times on the original compstatin peptide (Katragadda et al., 2006, J Med Chem 49 : 4616-4622; WO2007 / 062249).

Although the previous optimization steps have been based on combinatorial selection studies, solution structures, and computational models (Chiu et al., 2008, Chem Biol Drug Des 72: 249-256; Mulakala et al., 2007, Bioorg Med Chem 15: 1638-1644; Ricklin & Lambris, 2008, supra), the recent publication of a compstatin co-crystal structure in complex with the C3c complement fragment (Janssen et al., 2007, J Biol Chem 282: 29241- 29247; WO2008 / 153963) represents an important milestone to initiate rational optimization. The crystal structure revealed a surface link site at the interface of the macroglobulin (MG) domains 4 and 5 of C3c and showed that 9 of the 13 amino acids were directly involved in the bond, either through

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hydrogen bonds or hydrophobic effects. Compared to the structure of the compstatin peptide in solution (Morikis et al., 1998, supra), the form of compstatin binding underwent a conformational change, with a change in the location of the p-turn of residues 5-8 a 8-11 (Janssen et al., 2007, supra; WO2008 / 153963).

In view of the above, it is clear that the development of modified or mimetic compstatin peptides with even greater activity would constitute a significant advance in the art.

SUMMARY OF THE INVENTION

The present invention provides analogs and mimetics of the peptide that inhibits complement, compstatin, ICVVQDWGHHRCT (cyclic C2-C12); SEQ ID NO: 1), which have enhanced complement inhibition activity compared to compstatin.

One aspect of the invention has a compound comprising a modified compstatin peptide (ICVVQDWGHHRCT (cyclic C2-C12); SEQ ID NO: 1), in which the Gly at position 8 is modified to restrict the conformation of the peptide skeleton in that location. In one embodiment, the skeleton is restricted by replacing the Gly with N-methyl Gly. The peptide may be further modified by one or more of: replacement of His at position 9 with Ala; Val replacement at position 4 with Trp or a Trp analog; Trp replacement at position 7 with a Trp analog; acetylation of the N-terminal residue; and replacement of Thr at position 13 with Ile, Leu, Nle, N-methyl Thr or N-methyl Ile. In particular embodiments, the Trp analog in position 4 is 1-methyl Trp or 1-formyl Trp and the analog of Trp in position 7, if any, is a halogenated Trp.

Certain embodiments have a compstatin analog comprising a peptide having a sequence of SEQ ID NO: 2, which is:

Xaa1 - Cys - Val - Xaa2 - Gin - Asp - Xaa3 - Gly - Xaa4 - His - Arg - Cys - Xaa5 (cyclic C2-C12) where Gly in position 8 is modified to restrict the conformation of the peptide skeleton in that location, and where:

Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide comprising Gly-Ile;

Xaa2 is Trp, halogenated Trp, Trp comprising a lower alkoxy or alkanoyl substituent in the

position 1;

Xaa3 is halogenated Trp or Trp; Xaa4 is His, Ala, Phe or Trp; Y

Xaa5 is Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile, wherein a carboxy terminal -OH of any of

Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile is optionally replaced by -NH2.

In certain embodiments, Xaa2 participates in a non-polar interaction with C3. In other embodiments, Xaa3 participates in a hydrogen bond with C3. In several embodiments, the Xap2 Trp analog is a halogenated tryptophan, such as 5-fluoro-1-tryptophan or 5-fluoxo-1-tryptophan. In other embodiments, the Trp analog in Xaa2 comprises a lower alkoxy or lower alkyl substituent in the 5-position, for example, 5-methoxytryptophan or 5-methyltriptophan. In other embodiments, the Trp analog in Xaa2 comprises a lower alkyl or lower alkenoyl substituent at position 1, with exemplary embodiments comprising 1-methyltriptophan or 1-formyltriptophan. In other embodiments, the Xap3 Trp analog is a halogenated tryptophan such as 5-fluoro-1- tryptophan or 6-fluoro-1-tryptophan. In particular embodiments, Xaa2 is 1-methyltryptophan or 1-formyltriptophan and Xaa3 optionally comprises 5-fluoro-1-tryptophan.

In certain embodiments, Xaa1 is Ac-Ile, Xaa2 is 1-methyl-Trp or 1-formyl-Trp, Xaa3 is Trp, Xaa4 is Ala, and Xaa5 is Thr, Ile, Leu, Nle, N-methyl Thr or N- methyl Ile, In particular, Xaa5 can be Ile, N-methyl Thr or N-methyl He. In particular, the compstatin analog comprises any of SEQ ID NOS: 5, 7, 8, 9, 10 or 11.

In some embodiments, the compound comprises a peptide produced by the expression of a polynucleotide encoding the peptide. In other embodiments, the compound is produced at least in part by peptide synthesis. A combination of synthetic methods can also be used.

Another aspect of the invention has a compound of any of the preceding claims, further comprising an additional component that extends the in vivo retention of the compound. The additional component is polyethylene glycol (PEG) in one embodiment. The additional component is a small albumin binding molecule in another embodiment. In another embodiment, the additional component is an albumin binding peptide. The albumin binding peptide may comprise the sequence RLIEDICLPrWgCLWEDD (SEQ ID NO: 14). Particular embodiments comprise any of SEQ ID NOS: 5, 7, 8, 9, 10 or 11 linked to the albumin binding peptide. Optionally, the compound and the albumin binding peptide are separated by a spacer. The spacer can be a polyethylene glycol (PEG) molecule, such as mini-PEG or mini-PEG 3.

Another aspect of the invention presents the compound that inhibits complement activation, comprises a non-peptide mimetic or partial peptide of any of SEQ ID NOS: 5, 7, 8, 9, 10 or 11, wherein the

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compound binds C3 and inhibits complement activation with at least 500 times greater activity than the peptide comprising SEQ ID NO: 1 under equivalent test conditions.

The compstatin analogs, conjugates and mimetics of the invention are of practical utility for any purpose for which the same compstatin is used, as is known in the art and described in greater detail herein. Certain of these uses involve the formulation of the compounds in pharmaceutical compositions for administration to a patient. Such formulations may comprise pharmaceutically acceptable salts of the compounds, as well as one or more pharmaceutically acceptable diluents, carriers, excipients and the like, as would be within the skill of the person skilled in the art.

Various features and advantages of the present invention will be understood with reference to the detailed description, drawings and examples that follow.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions:

Various terms related to the methods and other aspects of the present invention are used throughout the specification and claims. These terms should be given their ordinary meaning in the art unless otherwise specified. Other specifically defined terms should be interpreted in a manner consistent with the definition provided herein.

The term "approximately" as used herein when referring to a measurable value as a quantity, a temporary duration, and the like, is intended to cover variations of ± 20% or ± 10%, in some embodiments ± 5% , in some embodiments ± 1%, and in some embodiments ± 0.1% of the specified value, since such variations are appropriate for making and using the disclosed compounds and compositions.

The term "compstatin" as used herein refers to a peptide comprising SEQ ID NO: 1, ICVVQDWGHHRCT (C2-C12 cyclic). The term "compstatin analog" refers to a modified compstatin comprising natural and non-natural amino acid substitutions, or amino acid analogs, as well as modifications within or between several amino acids, as described in greater detail herein, and as Knows in the art. When reference is made to the location of particular amino acids or analogs within compstatin or compstatin analogs, those locations are sometimes referred to as "positions" within the peptide, with positions numbered from 1 (Ile in compstatin) to 13 (Thr in compstatin). For example, the Gly residue occupies "position 8".

The terms "pharmaceutically active" and "biologically active" refer to the ability of the compounds of the invention to bind with C3 or fragments thereof and inhibit complement activation. This biological activity can be measured by one or more of the various assays recognized in the art, as described in greater detail herein.

As used herein, "alkyl" refers to an optionally substituted linear, branched or cyclic saturated hydrocarbon having from about 1 to about 10 carbon atoms (and all combinations and sub-combinations of ranges and specific numbers of carbon atoms in it), with about 1 to about 7 carbon atoms being preferred. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl, cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl. The term "lower alkyl" refers to an optionally substituted linear, branched or cyclic saturated hydrocarbon having from about 1 to about 5 carbon atoms (and all combinations and sub-combinations of ranges and specific numbers of carbon atoms therein) . Lower alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl, isopentyl and neopentyl.

As used herein, "halo" refers to F, Cl, Br or I.

As used herein, "alkanoyl," which can be used interchangeably with "acyl," refers to an optionally substituted linear or branched aliphatic acyl residue having from about 1 to about 10 carbon atoms (and all combinations. and sub-combinations of intervals and specific numbers of carbon atoms therein), with from about 1 to about 7 carbon atoms being preferred. Alkanoyl groups include, but are not limited to, formyl, acetyl, propionyl, butyryl, isobutyrylopentanoyl, isopentanoyl, 2-methyl-butyryl, 2,2-dimethylpropionyl, hexanoyl, heptanoyl, octanoyl, and the like. The term "lower alkanoyl" refers to an optionally substituted linear or branched aliphatic acyl residue having from about 1 to about 5 carbon atoms (and all combinations and sub-combinations of ranges and specific numbers of carbon atoms therein). Lower alkanoyl groups include, but are not limited to, formyl, acetyl, n-propionyl, iso-propionyl, butyryl, isobutyryl, pentanoyl,

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iso-pentanoyl, and the like.

As used herein, "aryl" refers to an optionally substituted mono- or bicyclic aromatic ring system having from about 5 to about 14 carbon atoms (and all combinations and sub-combinations of intervals and specific numbers of atoms carbon in it), with about 6 to about 10 carbons being preferred. Non-limiting examples include, for example, phenyl and naphthyl.

As used herein, "aralkyl" refers to alkyl radicals that carry an aryl substituent and have from about 6 to about 20 carbon atoms (and all combinations and sub-combinations of ranges and specific numbers of carbon atoms in the same), with about 6 to about 12 carbon atoms being preferred. Aralkyl groups may be optionally substituted. Non-limiting examples include, for example, benzyl, naphthylmethyl, diphenylmethyl, triphenylmethyl, phenylethyl and diphenylethyl.

As used herein, the terms "alkoxy" and "alkoxy" refer to an optionally substituted alkyl-O-group in which alkyl is as defined above. Exemplary alkoxy and alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy and heptoxy, among others.

As used herein, "carboxy" refers to a group -C (= O) OH.

As used herein, "alkoxycarbonyl" refers to a group -C (= O) O-alkyl, where alkyl is as defined above.

As used herein, "aroyl" refers to a group -C (= O) -aryl, in which aryl is as defined above. Exemplary aroyl groups include benzoyl and naphthoyl.

Typically, the substituted chemical fractions include one or more substituents that replace hydrogen at selected locations in a molecule. Exemplary substituents include, for example, halo, alkyl, cycloalkyl, aralkyl, aryl, sulfhydryl, hydroxyl (-OH), alkoxy, cyano (-CN), carboxyl (-COOH), acyl (alkanoyl: -C (= O) R ); -C (= O) O-alkyl, aminocarbonyl (-C (= O) NH2), aminocarbonyl -N-substituted (-C (= O) NHR "), CF3, CF2CF3, and the like. In relation to additional substituents , each R '' fraction can be, independently, any of H, alkyl, cycloalkyl, aryl, or aralkyl, for example.

As used herein, "amino acid L" refers to any of the naturally occurring alpha-levogyric amino acids normally present in proteins or the alkyl esters of those alpha amino acids. The term "amino acid D" refers to alpha dextrogyric amino acids. Unless otherwise specified, all amino acids referred to herein with L amino acids.

"Hydrophobic" or "non-polar" are used as synonyms herein, and refer to any intero-molecular interaction not characterized by a dipole.

"PEGylation" refers to the reaction in which at least a fraction of polyethylene glycol (PEG), regardless of its size chemically binds to a protein or peptide to form a PRG-peptide conjugate. "PEGylated" means that at least a fraction of PEG, regardless of size, chemically binds to a peptide or protein. The term PEG is generally accompanied by a numerical suffix indicating the approximate average molecular weight of the PEG polymers; For example, PEG-8,000 refers to polyethylene glycol having an average molecular weight of approximately 8,000.

As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the original compound is modified by making salts of acids or bases thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids, residues such as amines, alkaline or organic salts of acidic residues such as carboxylic acids; and the like Thus, the term "acid addition salt" refers to the corresponding salt derivative of an original compound that has been prepared by the addition of an acid. Pharmaceutically acceptable salts include conventional salts or quaternary ammonium salts of the original compound formed, for example, from inorganic or organic acids. For example, such conventional salts include, but are not limited to, those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroximaleic, phenylacetic, glutamic, benzoic, salicylic, sulphanilic, 2-acetoxybenzoic acids, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, isetionic oxalic, and the like. Certain acidic or basic compounds of the present invention may exist as zwitterions. It is contemplated that all forms of the compounds, including free acid, free base, and zwitterions, are within the scope of the present invention.

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Description:

In accordance with the present invention, information on the biological and physicochemical characteristics of compstatin that binds with C3 has been employed to design compstatin peptides with significantly improved activity compared to the original compstatin peptide. In some embodiments, analogs have at least 500 times more activity than compstatin. In other embodiments, the analogs have 350-, 400-, 450-, 500-, 550-, 600-fold or more activity than compstatin, compared to the tests described in the examples.

Compstatin analogs synthesized according to the above approaches have shown that they have improved activity compared to the original peptide, i.e. up to about 99 times (Mallik, B. et al, 2005, supra; WO2004 / 026328), and up to about 264 times (Katragadda et al., 2006, supra; WO2007 / 062249). The analogs according to the present invention demonstrate improved activity by modification in a position of compstatin not used so far, and can impart enhanced activity to compstatin or any analogue currently described. The analogs of the present invention therefore possess even greater activity than either the original peptide or analogs thereof produced to date, as demonstrated by in vitro assays as shown in the figures and in the Examples described herein.

The following table shows the amino acid sequence and complement inhibitory activities of selected exemplary analogs with significantly improved activity on compstatin (Ic [CVVQDWGHHRC] T; SEQ ID NO: 1). Selected analogs are referred to by specific modifications of the designated positions (1-13) compared to a potent compstatin analogue (Ac-Ic [CV (1 'MeW) QDWGAHRC] T-NH2, SEQ ID NO: 4, also referred to as peptide 14 in Example 1) which was described in WO2007 / 062249. The peptides of SEQ ID NOS: 5 and 7-11 (also referred to as peptides 15 and 17-21 in Example 1) are representative of modifications made in accordance with the present invention, resulting in significantly more potent compstatin analogs.

Exemplary compstatin analogs, IC50, and times of change in relation to SEQ ID NO: 4 (Ac-Ic [CV (1-MeW) QDWGAHRC] T-NH2), IC50 of 206 nM):

 SEQ ID NO:

 Pept No. Xaa8 Xaa13 Sequence IC50 (nM) Times of change

 5

 15 Sar * Thr Ac-Ic [CV (1-MeW) QDW (N-MeG) AHRC] T-NH2 159 1.30

 7

 17 Sar Ile Ac-Ic [CV (1-MeW) QDW (N‘MeG) AHRC] I-NH2 92 2.24

 8

 18 Sar Leu Ac-Ic [CV (1-MeW) QDW (N-MeG) AHRC] L-NH2 108 1.91

 9

 19 Sar Nle Ac-Ic [CV (1-MeW) QDW (N-MeG) AHRC] (Nle) -NH2 109 1.90

 10

 20 Sar (NMe) Thr Ac-Ic [CV (1-MeW) QDW (N-MeG) AHRC] (N-MeT) -NH2 86 2.40

 eleven

 21 Sar (NMe) Ile Ac-Ic [CV (1-MeW) QDW (N-MeG) AHRC] (N-MeI) -NH2 62 3.32

 * Sar =

 N-ME Gly

A modification according to the present invention comprises restriction of the peptide skeleton at position 8 of the peptide. In a particular embodiment, the skeleton is restricted by replacing glycine in position 8 (Gly8) with N-methyl glycine. Reference is made to exemplary peptides 8 and 15 as discussed in Example 1.

Without wishing to be bound or limited by theories, it should be noted that N-methylation can affect a peptide in various ways. First, the potential hydrogen donor is replaced with a methyl group, which cannot form a nitrogen bond. Second, the N-methyl group is a weak electron donor which means that it can slightly increase the basicity of the adjoining carbonyl group. Third, the size of the N-methyl group could cause steric restriction. Finally, N-methylation can change the trans / cis population of the amide bond, thus changing the local peptide conformation in a similar way to proline.

The increased activity of [Trp (Me) 4Gly (N-Me) 8Ala9] -Ac-compstatin (SEQ ID NO: 5; peptide 15) is a marked improvement compared to the previously more active analog, [Trp (Me ) 4Gly8Ala9] -Ac-compstatin (SEQ ID NO: 4; peptide 14). The N-methylation of Gly8 probably improves the recognition of the target and the stability of the complex by reinforced p-type twist, increased local skeleton constraints and improved hydrophobic interactions involving the Trp7 side chain.

In particular embodiments, the modification at position 8 is supplemented by an additional modification comprising replacing Thr at position 13 with Ile, Leu, Nle (norleucine), N-methyl Thr or N-methyl Ile. Reference is made to the exemplary peptides 16, 17, 18, 19, 20 and 21 (SEQ ID NOS: 6, 7, 8, 9, 10 and 11) as discussed in Example 1. Again, without wishing to be limited or bound by theory, it was discovered that the replacement of Thr with hydrophobic Ile is beneficial. Similar effects observed for the two isomers of Ile (i.e., Leu and Nle)

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They suggest that physicochemical and steric properties, rather than specific contacts, may be responsible for this improvement. However, a more distinctive improvement in affinity and activity was observed after N-methylation of the skeleton of both Thr13 and Ile13. While the improvements observed may have been the result of increased skeletal constraints, and therefore minor conformational entropic penalties after bonding, it is also the case that the nature of the residue in position 13 may additionally influence the formation and stabilization of active conformations, either sterically or through the formation of intramolecular hydrophobic contacts.

The modifications described above at position 8 and position 13 can be combined with other compstatin modifications shown above to improve activity, to produce peptides with significantly improved complement inhibitory activity. For example, acetylation of the term N typically increases the inhibitory activity of compstatin complement and its analogs. Accordingly, the addition of an acyl group in the amino terminus of the peptide, including but not limited to N-acetylation, is a preferred embodiment of the invention, particularly useful when the peptides are synthetically prepared. However, it is sometimes advantageous to prepare the peptides by expression of a nucleic acid molecule encoding the peptide in a prokaryotic or eukaryotic expression system, or by transcription and translation in vitro. For these embodiments, the term N of natural origin can be used.

As another example, it is known that the substitution of Ala by His at position 9 improves the activity of compstatin and is a preferred modification of the peptides of the present invention as well. It has also been determined that the substitution of Tyr with Val at position 4 can lead to a modest improvement in activity (Klepeis et al., 2003, J Am Chem Soc 125: 8422-8423).

It has also been disclosed in WO2004 / 026328 and WO2007 / 0622249 that Trp and certain analogs of Trp in position 4, as well as certain analogues of Trp in position 6, especially combined with Ala in position 9, produce activity many times greater than that of the compstatin. These modifications are also advantageously used in the present invention as well.

In particular, peptides comprising 5-fluoro - / - tryptophan or 5-methoxy-, 5-methyl- or 1-methyl-tryptophan, or 1-formyl-tryptophan at position 4 have been shown to have activity 31-264 times greater than that of compstatin. Particularly preferred with 1-methyl and 1-formyl tryptophan. It is believed that a hydrogen bond 'N'-mediated by indole is not necessary at position 4 for the binding and activity of compstatin. The absence of this hydrogen bond or reduction of the polar character by replacing hydrogen with lower alkyl, alkanoyl or indole nitrogen at position 4 enhances the binding and activity of the compstatin. Without wishing to be limited by any particular theory or mechanism of action, it is believed that a hydrophobic interaction or effect at position 4 reinforces the interaction of compstatin with C-. Accordingly, the modifications of Trp at position 4 or position 7 of Trp analogs that maintain or enhance the hydrophobic interaction mentioned above are contemplated in the present invention as an advantageous modification in combination with the modifications at positions 8 and 13 as described above. Such analogs are well known in the art and include, but are not limited to analogs exemplified herein, as well as unsubstituted or alternatively substituted derivatives thereof. Examples of suitable analogs can be found by reference in the following publications, and many others Beene, et al., 2002, Biochemistry 41: 10262-10269 (which describes, among other Trp analogs, individually and multi-halogenated); Babitzky & Yanofsky, 1995, J. Biol. Chem. 270: 1245212456 (which describes, among others, methylated and halogenated Trp and other analogs of Trp and indole); and U.S. Pat. 6,214,790. 6,169,057. 5,776,970. 4,870,097. 4,576,750 and 4,299,838. Trp analogs can be introduced into the compstatin peptide by expression in vitro or in vivo, or by peptide synthesis, as is known in the art.

In certain embodiments, Trp at position 4 of the compstatin is replaced with an analog comprising a 1-alkyl substituent, more particularly a lower alkyl substituent (eg, C1-C5) as defined above. These include, but are not limited to, N (a) methyl tryptophan, N (a) formyl tryptophan and 5-methyltriptophan. In other embodiments, Trp at position 4 of the compstatin is replaced with an analog comprising a 1-alacanoyl substituent, more particularly a lower alkanoyl substituent (eg, C1-C5) as defined above. In addition to the analogs specified, these include but are not limited to 1-acetyl-L-tryptophan and L-p-homotriptophan.

In WO2007 / 0622249 it was reported that the incorporation of 5-fluoro - / - tryptophan at position 7 in compstatin increased the enthalpy of the interaction between compstatin and C3, in relation to wild type compstatin, while incorporation of 5-fluoro-tryptophan at position 4 in the compstatin decreased the enthalpy of this interaction. Accordingly, the modifications of Trp at position 7, as described in WO2007 / 062224, are contemplated as useful modifications in combination with the modifications at positions 8 and 13 as described above.

The modified compstatin peptides of the present invention can be prepared by various synthetic methods of peptide synthesis by mediating the condensation of one or more amino acid residues, in accordance with conventional peptide synthesis methods. For example, peptides are synthesized according to

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Standard solid phase methodologies, as can be performed in an Applied Biosystems model 431A peptide synthesizer (Applied Biosystems, Foster City, Calif.), according to the manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase or liquid phase methodologies, are well known to those skilled in the art. During the course of peptide synthesis, branched chain amino and carboxyl groups can be protected / deprotected as necessary, using commonly known protecting groups. An example of a suitable synthetic peptide method is set forth in Example 1. Modification using alternative protecting groups for peptides and peptide derivatives will be apparent to those skilled in the art.

Alternatively, certain peptides of the invention may be produced by expression in a suitable prokaryotic or eukaryotic system. For example, a DNA construct can be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E.coli) or a yeast cell (such as Saccharomyces cerevisiae), or in a baculovirus vector for expression in a cell. of insect or viral vector for expression in a mammalian cell. Such vectors comprise the regulatory elements necessary for the expression of the DNA in the host cell, positioned in such a way as to allow the expression of the DNA in the host cells. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.

Peptides can also be produced by the expression of a nucleic acid molecule in vitro or in vivo. A DNA construct that encodes a concatamer of the peptides, the upper limit of the concatamer being dependent on the expression system used, can be introduced into an in vivo expression system. After the concathemer has been produced, cleavage between the C-terminal Asn and the next N-terminal G is achieved by exposure of the hydrazine polypeptide.

Peptides produced by gene expression in a recombinant prokaryotic or eukaryotic system can be purified according to methods known in the art. A combination of gene expression and synthetic methods can also be used to produce compstatin analogs. For example, an analogue can be produced by gene expression and subsequently subjected to one or more post-translational synthetic processes, for example to modify the term N- or C- or to cycle the molecule.

Advantageously, peptides incorporating unnatural amino acids, for example, methylated amino acids, can be produced by expression in vivo in a suitable prokaryotic or eukaryotic system. For example, methods such as those described by Katragadda & Lambris (2006, Protein Expression and Purification 47: 289-295) to introduce unnatural Trp analogs into compstatin by expression in E. coli auxotrophs can be used to introduce N- amino acids methylated or other unnatural in selected positions of compstatin.

The structure of compstatin is known in the art, and the structures of the preceding analogues are determined by similar means. Once a particular desired conformation of a short peptide has been proven, the methods for designing a peptide or peptidomimetic to adjust with that conformation are well known in the art. Of particular relevance to the present invention, the design of the peptide analogs can be further refined considering the contribution of several side chains of amino acid residues, as discussed above (ie, for the effect of functional groups or for steric considerations) .

It will be appreciated by those skilled in the art that a peptide imitator can serve equally well as a peptide for the purpose of providing the specific skeleton conformation and side chain functionalities required for binding with C3 and inhibiting complement activation. Accordingly, it is contemplated that it is within the scope of the present invention to produce compounds that bind with C3, which inhibit complement by the use of naturally occurring amino acids, amino acid derivatives, analogs or non-amino acid molecules capable of binding to form the conformation of the appropriate skeleton. A non-peptide analog, or an analog comprising peptide and non-peptide components, is sometimes referred to herein as a "peptidomimetic" or "isostatic mimetic", to designate substitutions or derivations of the peptides of the invention, which possesses the same. characteristics and / or other conformational functionalities of the skeleton, to be sufficiently similar to the exemplified peptides to inhibit complement activation.

The use of peptidomimetics for the development of high affinity peptide analogs is well known in the art (see for example, Vagner et al., 2008, Curr. Opin. Chem. Biol. 12: 292-296; Robinson et al. , 2008, Drug Disc. Today 13: 944-951). Assuming rotational restrictions similar to those of amino acid residues within a peptide, analogs comprising non-amino acid fractions can be analyzed, and their conformational motifs verified, by a variety of computational techniques that are well known in the art.

The modified compstatin peptides of the present invention can be modified by the addition of polyethylene glycol (PEG) components to the peptide. As is well known in the art, PEGylation can increase the half-life of therapeutic peptides and proteins in vivo. In one embodiment, the PEG has a weight

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Average molecular from about 1,000 to about 50,000. In another embodiment the PEG has a weight

Average molecular from about 1,000 to about 20,000. In another embodiment the PEG has a weight

Average molecular from about 1,000 to about 10,000. In an exemplary embodiment, the PEG has

an average molecular weight of approximately 5,000. The polyethylene glycol can be a branched or linear chain, and

preferably it is a linear chain.

The compstatin analogs of the present invention can be covalently linked to PEG by a linkage group. Such methods are well known in the art. (Revised in Kozlowski A. et al. 2001, BioDrugs 15: 419-29; see also, Harris JM and Zalipsky S, eds. Poly (ethylene glycol), Chemistry and Biological Applications, ACS Symposium Series 680 (1997)). Non-limiting examples of acceptable linkage groups include an ester group, an amide group, an imide group, a carbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, a succinimide group (including without limitation, succinimidyl succinate (SS), succinimidyl propionate (SPA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA) and N-hydroxy succinimide (NHS)), an epoxy group, an oxycarbonylimidazole group (including, without limitation, carbonyldimidazole (CDI)), a nitro phenyl group (including, without limitation, nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), a triesylate group, an aldehyde group, an isocyanate group, a vinyl sulfone group, a tyrosine group, a cysteine group, a histidine group or a primary amine In certain embodiments, the linkage group is a succinimide group. In one embodiment, the linkage group is NHS.

The compstatin analogs of the present invention can alternatively be directly coupled with PEG (ie, without a linkage group) by an amino group, a sulfhydryl group, a hydroxyl group or a carboxyl group. In one embodiment, the PEG is coupled with a lysine residue added to the C term of compstatin.

As an alternative to PEGylation, in vivo clearance of peptides can also be reduced by linking the peptides with certain other molecules or peptides. For example, certain albumin binding peptides show an unusually long half-life of 2.3 h when injected intravenously into rabbits (Dennis et al., 2002, J Biol Chem. 277: 35035-35043). Such a peptide, fused with the anti-tissue factor Fab of D3H44 allowed the Fab to bind with albumin while retaining the ability of the Fab to bind the tissue factor (Nguyen et al., 2006, Protein Eng Des Sel. 19: 291-297.). This interaction with albumin resulted in significantly reduced in vivo clearance and extended half-life in mice and rabbits, when compared to Fab D3H44 of the wild type, comparable to those observed for PEGylated Fab molecules, immunoadhesins, and albumin fusions. WO2007 / 062249 describes a compstatin analog fused with an albumin binding peptide (ABP) and reports that the fusion protein is active in inhibiting complement activation. However, the synthesis was long and the production of the fusion product was less than desired. Example 2 of the present exposes improved synthesis strategies that use an ABP as well as a small albumin binding molecule (ABM), and optionally employ a spacer between the components. These procedures resulted in the production of ABP- and ABM-compstatin analog conjugates capable of inhibiting complement activation and also showing extended in vivo survival. In fact, ABP was able to improve the half-life of a compstatin analogue 21 times without significantly compromising its inhibitory activity. Thus, such conjugates allow systemic administration of the inhibitor without infusion.

The complement inhibitory activity of compstatin analogs, peptidomimetics and conjugates can be tested by a variety of assays known in the art. In one embodiment, the test described in Example 1 is used. A non-exhaustive list of other tests is set forth in U.S. Pat. 6,319,897, WO99 / 13899 WO2004 / 026328 and WO2007 / 06224 including, but not limited to, (1) peptide bonding with C3 and C3 fragments; (2) various hemolytic assays; (3) measurement of cleavage mediated by C3 C3 convertase; and (4) measurement of excision of Factor B by Factor D.

The peptides and peptidomimetics described herein are of practical utility for any purpose for which the same compstatin is used, as is known in the art. Such uses include, but are not limited to: (1) inhibiting complement activation in the serum, tissues or organs of a patient (human or animal), which may facilitate the treatment of certain diseases or conditions, including but not limited to , age-related macular degeneration, rheumatoid arthritis, spinal cord injury, Parkinson's disease, Alzheimer's disease, cancer and respiratory disorders such as asthma, chronic obstructive pulmonary disease (COPD), allergic inflammation, emphysema, bronchitis, bronchiectasis, fibrosis cystic, tuberculosis, pneumonia, respiratory distress syndrome (RDS - neonatal and adult), rhinitis and sinusitis; (2) inhibit complement activation that occurs during the transplantation of cells or organs, or in the use of artificial organs or implants (for example, by coating or otherwise treating cells, organs, artificial organs or implants with a peptide of the invention); (3) inhibit complement activation that occurs during extracorporeal bypass of physiological fluids (blood, urine) (for example, by coating the tubing by which the fluids are derived with a peptide of the invention); and (4) selection of small molecule libraries to identify other inhibitors of compstatin activation (eg, high performance tests in liquid-solid phase) designed to measure the ability of a test compound to compete with an analogue of compstatin to bind with C3 or a fragment of C3).

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To implement one or more of the utilities mentioned above, another aspect of the invention presents pharmaceutical compositions comprising the compstatin analogs or conjugates described and exemplified herein. Such a pharmaceutical composition may consist of the active ingredient only, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination thereof. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, as in combination with a physiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions can be prepared by any method known or developed hereafter in the art of pharmacology. In general, such preparatory methods include the step of carrying the active ingredient in association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product in a single dose unit or Multi-dose desired.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which a complement inhibitor can be combined and which, after combination, can be used to administer the complement inhibitor to a mammal.

The following examples are provided to describe the invention in greater detail. It is intended to illustrate, not limit, the invention.

Example 1

A mono-Na-methylation scan was performed on [Tyr4Ala9] -Ac-compstatin (Ac-Ic [CVYQDWGAHRC] T-NH2; SEQ ID NO: 3). Based on the test results of these analogs, selective N-methylation and substitution at position 13 in [Trp (Me) 4Ala9] -Ac-compstatin (Ac-Ic [CV (1-MeW) QDWGAHRC] T was performed -NH2; SEQ ID NO: 4). Selected analogs were further characterized using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Dynamic molecular simulations (MD) were also performed to investigate possible mechanisms for the observed increase in affinity.

Materials and methods:

Abbreviations Ac, acetyl group; Acm, acetamidomethyl; Boc, tert-butoxycarbonyl; CHARMM, Chemistry at Harvard Macromolecular Mechanics; DCM, dichloromethane; DIC, 1,3-diisopropylcarbodiimide; DIPEA, N, N-diisopropylethylamine; DMF, N, N-dimethylformamide; ELISA, enzyme-linked immunoabsorbent assay; ESI, electrospray ionization; Fmoc, 9-fluorenylmethoxycarbonyl; HOAt, 1-hydroxy-7-aza-benzotriazole; ITC, isothermal titration calorimetry; MALDI, matrix-assisted laser desorption ionization; MBHA, 4-methylbenzhydrylamine; MOE, molecular operating environment; NAMD, nanoscale molecular dynamics; Nle, L-norleucine; NMP, N-methylpyrrolidinone; RMSD, mean square deviation; SPR, surface plasmon resonance; TIPS, triisopropylsilane; Trt, trityl.

Chemical products. Low-loading Rink MBHA amide resin and the following Fmoc amino acids were obtained from Novabiochem (San Diego, CA): Ile, Cys (Acm), Val, Tyr (tBu), Gln (Trt), Asp (OtBu), Trp (Boc), Gly, Sar, Ala, MeAla, His (Trt), Arg (Pmc), Melle, Nle, Phe, and Thr (tBu). DIC and Fmoc-Trp (Me) -OH were purchased from AnaSpec (San Jose, CA). HOAt was acquired from Advanced ChemTech (Louisville, KY). NMP and DCM were obtained from Fisher Scientific (Pittsburgh, PA). All other chemical reagents for synthesis were obtained from Sigma-Aldrich (St. Louis, MO) and used without further purification.

Synthesis and purification of peptides. All peptides were synthesized manually by Fmoc solid phase methodology using DIC and HOAt as coupling reagents. When N-methylated amino acids were not commercially available, Na-methylation was performed using the optimized methodology reported by Biron et al. (2006, J Peptide Sci 12: 213-219). The following procedures were used for the synthesis of the linear peptides, Rink MBHA amide resin (294 mg, 0.34 mmol / g) was placed in a 10 ml HSW polypropylene syringe with frits at the bottom (Torviq, Niles , MI) and swelled in DCM (5 ml) for 30 minutes. After removal of the Fmoc protective group (25% piperidine in NMP, 5 ml, 5 and 10 min), the resin was washed four times with NMP (5 ml per wash) and DCM (5 ml per wash), and coupled the individual amino acids with the resin. For each coupling, 3 equivalents (3 mmol) of the amino acid, HOAt, and DIC were used, with 10 min preactivation in NMP. All couplings were made for 1 hour and monitored by the Kaiser test or the chloranil test. In case of a positive test result, the coupling was repeated until a negative test result was observed.

The N-terminal amino group was acetylated with 20 equivalents of acetic anhydride and 2 equivalents of DIPEA in 5 ml of DCM for 30 min. Linear peptides containing Cyc residues (Acm) were cycled in resin using thallium acetate in DMF / anisole (19: 1) at room temperature for 3 h. The resin was washed four times

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with DMF, DCM, and DCM / diethyl ether (1: 1) (every 5 ml per wash), and dried under vacuum for 4 h. The peptides were cleaved from resin with a mixture of 95% TFA, 2.5% water, and 2.5% TIPS for 3 hours. After evaporation of the TFA in vacuo, the peptides were precipitated and washed three times with 30 ml of cold diethyl ether per wash. The liquid was separated from the solid by centrifugation and decanted. The crude peptides were air dried and dissolved in acetonitrile and 0.1% tFa in water (1: 3) before purification by preparative RP-HPLC (Vydac C18 column 218TP152022, Western Analytical Products, Murrieta, CA) and elution with a linear gradient of 15-50% acetonitrelo in 0.1% aqueous TFA solution for 35 min at a flow rate of 15 ml / min. Fractions containing the desired products were collected, concentrated, and lyophilized. The purified peptides were isolated in general yields of 10-15% and were> 95% pure as determined by analytical RP-HPLC (Phenomenex 00G-4041-E0 Luna 5m C18 100A column, 250x4.60 mm; Phenomenex, Torrance, CA ). The mass of each peptide was confirmed using ThermoQuest Finnigan LCQ Duo and Waters MALDI micro MX instruments.

C3 purification. C3 was purified from new human plasma obtained from the blood bank of the University of Pennsylvania Hospital. Briefly, the plasma was fractionated with 15% (w / v) PEG 3350, and the pellet was resuspended in 20 mM phosphate buffer, pH 7.8, and then subjected to anion exchange chromatography on a DEAE-HR column 40 (50 3 5 cm; Millipore Inc., Billerica, MA) with the same buffer. The proteins were eluted with 6 l of a linear gradient (15-70%) of 20 mM phosphate buffer, pH 7.8, containing 500 mM NaCl. The C3 was further purified on a Superdex 200 26/60 size exclusion column (Amersham Biosciences) and a Mono S column (Amersham Biosciences) to separate C3 from C3 (H2O).

Inhibition of complement activation. The ability of compstatin analogs to inhibit the activation of the classical complement pathway by ELISA was evaluated (Mallik et al., 2005, J Med Chem 48: 274-86). Briefly, the complement was activated in human serum using an antigen-antibody complex in the presence or absence of compstatin analogs, and the deposition of C-fragments on the surface of the plate was detected using a polyclonal HRP-conjugated anti-C3 antibody . The absorbance data obtained at 405 nm was translated in% inhibition, based on the absorbance corresponding to 100% activation of the supplements. The percent inhibition was plotted against the peptide concentration, and the resulting data set was adjusted to the logistic dose response function using Origin 7.0 software. The IC50 values were obtained from the adjusted parameters that produced the lowest value of x2. Each analog was tested at least three to seven times. The standard deviations were all within 30% of the average value.

ITC analysis. All ITC experiments were performed with the Microcal VP-ITC calorimeter (Microcal Inc., Northampton, MA), using protein concentrations of 1.8-5 pM of C3 in the cell and peptide concentrations of 40-100 pM of Individual compstatin analogues in the syringe. All titers were made in PBS (10 mM phosphate buffer with 150 mM NaCl, pH 7.4) at 25 ° C using multiple peptide injections of 2-7 pl each. The crude isotherms were corrected for the dilution heats by subtracting the peptide injections representing isotherms in the buffer. The resulting isotherms were adjusted to an individual site model site using Origin 7.0 software, and the model that produced the lowest value of x2 was considered appropriate for the respective data set. Gibbs free energy was calculated as AG = AH-TAS. Each experiment was repeated at least twice. The errors were within 20% of the average values.

SPR analysis. The kinetics of the interaction between C3b and each compstatin analog by SPR were analyzed on a Biacore 3000 instrument (GE Healthcare Corp., Piscataway, NJ) at 25 ° C using PBS-T (10 mM sodium phosphate, 150 mM NaCl , 0.005% Tween-20, pH / 4)) as the run buffer, as described above. Briefly, biotinylated C3b (30 pg / ml) was immobilized on a streptavidin coated detection chip, and a double serial dilution series (1 pM-500 pM) of each analog was injected for 2 min at 30 pl / min, with a dissociation phase of 5-10 min. The Trp (Me) 4] -Ac-compstatin peptide was included in each experimental series as an internal control and reference. Data analysis was performed using Scrubber (BioLogic Software, Campbell, Australia). The signals from an untreated flow cell and a set of blank buffer injections were subtracted to correct the effects of the buffer and injection artifacts. The processed biosensor data were adjusted globally to a 1: 1 Langmuir link model, and the equilibrium dissociation constant (Kd) was calculated from the equation Kd = kd / ka. Peptide solutions were injected in duplicate in each experiment, and each screening test was performed at least twice. The error of kd and ka was within 10% of the average values.

Simulation of molecular dynamics. All MD simulations were performed with the NAMD program (Phillips, et al., 2005, J. Comput. Chem. 26: 1781-1802) using the force field CHARMM27. For free compstatin analogs, the NMR structure (Morikis & Lambris, 2002, Biochem. Soc. Trans. 30: 1026-1036) (PDB code: 1A1P) was adopted to construct starting structures. Point mutations were introduced with the Molecular Operating Environment program (MOE, Chemical Computing Group, 2005). Mutated residues of compstatin analogues were minimized using CHARMM (Brooks et al., 1983, J. Comput. Chem. 4: 187-217) version c33b1, with the CHARMM27 parameter set (MacKerell et al., 1998, J Phys. Chem. B 102: 35863616), while harmonic restrictions were placed on the atoms of the skeleton. The C3c residues of the complement missing from the crystal structure were added using homology modeling and also minimized using CHARMM.

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Crystallographic water molecules were maintained in the PDB file, and the structures were solvated in periodic cubic boxes of TIP3P water molecules (Jorgensen et al., 1983, J. Chem. Phys. 79: 926-935). The distances between the edges of the water simulation box and the closest atom of the solutes were at least 10 A. Sodium and chloride counterions were then added using the VMD program (Humphrey et al., 1996, J Mol. Graphics 14: 33-38, 27-28) to maintain the electroneutrality of the systems.

The systems were first minimized in three consecutive steps, during which the protein was initially fixed and the water molecules were allowed to move during 10,000 gradient steps of the conjugate; then only the protein skeleton was fixed for 100,000 steps; finally, all atoms were allowed to move for an additional 10,000 steps. The particle mesh Ewald method (Darden et al., 1993, J. Chem. Phys. 98: 10089-10092) was used to treat long-range electrostatic interactions under periodic isolation conditions with a grid of approximately 1 point per A. Non-bound van der Waals interactions were switched gently for 3 A between 9 and 12 A. Bond lengths involving hydrogen atom bonds were restricted using SHAKE (Ryckaert et al., 1977, J. Comput. Phys. 23: 327-341). The time period for the entire MD simulation was 2 fs. The Nose-Hoover Langevin piston (Feller et al., 1995, J. Chem. Phys. 103: 4613-4621; Martyna et al., 1994, J. Chem. Phys. 101: 4177-4189) was used for the control pressure, with the piston period set at 20 fs and a piston decay of 100 fs. MD simulations at 100 ps were carried out at a constant volume, during which the systems were heated to 310 K in 30 K increments; a subsequent isothermal isobaric MD simulation was used for 20 ns and 5 ns to adjust solvent density without any restriction on all solute atoms for free compstatin analogs and complexes, respectively. Finally, the most energy structures were obtained. lowered trajectory files balanced by MD and were subsequently used in the entropy and structure contribution analysis.

Results:

Inhibition of complement activation. N-methylation of the skeleton was performed on a template of [Tyr4 Ala9] -Ac-compstatin (peptide 1; SEQ ID NO: 3) to generate analogs 2-13 (Table 1-1). Although peptide 1 was less potent than the current starting compound, [Trp (Me) 4 Ala9] -Ac-compstatin (peptide 14, SEQ ID NO: 4), it was chosen for the initial scan due to its lower synthesis cost . The ability of each peptide to inhibit complement activation by ELISA was then evaluated and compared with the activity of peptide 1 (Table 1-1). The most negative effect was observed for the N-methylation of Val3, Tyr4 and Ala9, which made peptides 3, 4, and 9 completely inactive. In contrast, N-methylation of Gly8 and THr13 produced peptides 8 and 13 with slightly increased potency (1.7 and 1.3 times, respectively). N-methylation in all other positions resulted in detectable inhibitory activity, still significantly reduced (Table 1-1).

Table 1-1 Inhibition of activation by classical complement pathway by Na-methylated analogs of [Tyr4 Ala9] - ____________________________ Ac-compstatin (peptide 1; SEQ ID NO: 3) ____________________________

 Peptide

 Sequence IC50 (CP, pM) IC50-change times3

 1 B

 Ac-I [CVYQDWGAHRC] T-NH2 (SID 3) 2.4 1

 2

 Ac-I [(NMe) CVYQDWGAHRC] T-NH2 7.5 0.3

 3

 Ac-I [C (NMe) VYQDWGAHRC] T-NH2 NA NA

 4

 Ac-I [CV (NMe) YQDWGAHRC] T-NH2 NA NA

 5

 Ac-I [CVY (NMe) QDWGAHRC] T-NH2 33 0.07

 6

 Ac-I [CVYQ (NMe ') DWGAHRC] T-NH2 44 0.06

 7

 Ac-I [CVYQD (NMe ') WGAHRC] T-NH2 25 0.1

 8

 Ac-I [CVYQDW (NMe ') GAHRC] T-NH2 1.43 1.7

 9

 Ac-I [CVYQDWG (NMe ') AHRC] T-NH2 NA NA

 10

 Ac-I [CVYQDWGA (NMe ') HRC] T-NH2 94 0.03

 eleven

 Ac-I [CVYQDWGAH (NMe ') RC] T-NH2 32 0.08

 12

 Ac-I [CVYQDWGAHR (NMe ') C] T-NH2 154 0.02

 13

 Ac-I [CVYQDWGAHRC] (NNMe) T-NH2 1.89 1.3

 Note: to in

 relation to peptide 1. NA: not active, b Data from Sahu et al. , 1996, J. Immunol. 157: 884-891.

We then apply the findings of the N-methylation scan to the current potent analog, Ac-I [CV (1_ MeW) QDWGAHRC] T-NH2 (SEQ ID NO: 4; also referred to herein as Trp (Me) 4 Ala9] - Ac-compstatin, peptide 14), and analogs synthesized with selective N-methylation and amino acid substitutions at positions 8 and 13 (peptides 15-23; Table 1-2). As previous studies had indicated limitations to replace side chains in position 8 (Morikis et al., 1998, Protein Sci. 7: 619-627; Furlong et al., 2000, Immunopharmacology

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48: 199-212), the modifications were restricted to the absence (Gly8) or presence of N-methylation (NMeGly8, that is, Sar8). On the contrary, the previous work showed that the N-terminal position 13 allowed more flexibility for substitutions and had even suggested a preference for Ile over Thr (Morikis & Lambris, 2002, Biochem. Soc. Trans. 30: 1026-1036). Therefore, we further investigate the importance of position 13 and design a series of Sar8 analogs to include various N-methylated, hydrophobic, or aromatic residues in this position. Consistent with the results of the N-methylation scan, the introduction of a single N-methyl group at position 8 (Sar8; peptide 15) increased the inhibitory power by 1.3 times (Table 1-3). Additionally, the replacement of Thr by Ile at position 13 led to a significant increase in both Gly8 and Sar8 peptides. However, none of the substitutions of Ile for Leu or Nle, nor the introduction of His or Phe produced any improvement over the analog Ile13. In contrast, the N-methylation of both Thr13 and Ile13 resulted in a significant increase in inhibitory activity (IC50 = 86 and 62 nM, respectively), generating the most potent compstatin analogs described so far.

Claims (17)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty 55 60 65 Claims 1. A compound comprising a modified compstatin peptide having SEQ ID NO: 1, which is ICVVQDWGHHRCT (cyclic C2-C12), wherein the Gly at position 8 is modified to restrict the confirmation of the peptide skeleton in that location, where the skeleton is restricted by replacing the Gly with N-methyl Gly. 2. The compound of claim 1, further comprising replacing His in position 9 with Ala. 3. The compound of claim 1 or claim 2, further comprising replacing Val in position 4 with Trp, 1-methyl Trp or 1-formyl Trp. 4. The compound of any one of claims 1-3 further comprising replacing Trp in position 7 with halogenated Trp. 5. The compound of any of claims 1-4, further comprising acetylation of the N-terminal residue. 6. The compound of any of claims 1-5, further comprising replacing Thr at position 13 with Ile, Leu, Nle, N-methyl Thr or N-methyl Ile. 7. A compstatin analog comprising a peptide having SEQ ID NO: 2, which is: Xaa1 - Cys - Val - Xaa2 - Gln - Asp - Xaa3 - Gly - Xaa4 - His - Arg - Cys - Xaa5 (cyclic C2-C12) where Gly in position 8 is modified to restrict the conformation of the peptide skeleton in that location replacing the Gly with N-methyl Gly, where: Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide comprising Gly-Ile; Xaa2 is Trp, halogenated Trp, Trp comprising a lower alkoxy or lower alkyl substituent at position 5, or Trp comprises a lower alkyl or lower alkanoyl substituent at position 1; Xaa3 is halogenated Trp or Trp; Xaa4 is His, Ala, Phe or Trp; Y Xaa5 is Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile, where a carboxy terminal -OH of any of Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile is optionally replaced by -NH2. 8. The compound of claim 7, wherein Xaa1 is Ac-Ile, Xaa2 is 1-methyl-Trp or 1-formyl-Trp, Xaa3 is Trp, Xaa4 is Ala, and Xaa5 is Thr, Ile, Leu, Nle , N-methyl Thr or N-methyl Ile. 9. The compound of claim 7 or claim 8, comprising any of SEQ ID NOS: 5, 7, 8, 9. 10 or 11. 10. The compound of any of the preceding claims, linked to an additional component that extends the in vivo retention of the compound. 11. The compound of claim 10, wherein the additional component is polyethylene glycol (PEG) or a small albumin binding molecule. 12. The compound of claim 10, wherein the additional component is an albumin binding peptide; optionally having SEQ ID NO: 14, which is: RLIEDICLPRWGCLWEDD. 13. The compound of claim 12, wherein the compound and the albumin binding peptide are separated by a spacer; optionally where the spacer is a polyethylene glycol molecule. 14. A pharmaceutical composition comprising the compound of any of the preceding claims and a pharmaceutically acceptable carrier. 15. A compound of any of the preceding claims for use in inhibiting complement activation. 16. The in vitro use of a compound of any one of claims 1 to 14 for inhibition of complement activation.